The long term goal of this project is to understand the basis of sequence-specific recognition of DNA by proteins. DNA recognition governs many metabolic activities including gene activation and repression, site-specific recombination and initiation of DNA replication. Understanding how these proteins work will provide a framework for the rational design of new protein specificities that may have medical and pharmacological applications. The particular protein of study is the thermophilic restriction endonuclease TagI. A model has been proposed for TagI recognition of its cognate sequence. Further refinement of this model will involve characterization of wild type enzyme and mutants by genetic and biochemical means, isolation of putative mutants with altered sequence specificity, and isolation and characterization of an isoschizomer of TagI. Wild type endonuclease has been overproduced and purified as will a "canonical site nicking" mutant and an "allosteric activation" mutant. These will be characterized for sequence specific binding, dissociation, canonical and "star" site cleavage using a variety of DNA oligonucleotide and plasmid substrates. In collaboration with Dr. John Anderson, purified TagI will be co-crystallized with its cognate oligonucleotide for determination of its three dimensional structure. Mutants will be generated using two-codon insertion mutagenesis, oligonucleotide directed mutagenesis, and saturation mutagenesis. Two in vivo screens will be developed and may help identify mutants with altered sequence specificity. These mutants should help define amino acid residues involved in sequence-specific hydrogen bonds to the major groove, other sequence-specific contacts, allosteric activation of enzyme activity, and protein subunit interactions. The properties and sequence of an isoschizomer that recognizes DNA with similar structural motifs as TagI will be determined. The amino acid sequence of the isoschizomer might point to regions of functional homology in TagI necessary for sequence specific DNA recognition.